Part:BBa_K1497030

Scaffold (S-S)

The scaffold protein S1S1 (S-S) was constructed by the insertion of a SH3 BBa_K1497025 domain behind another SH3 domain. This was done by using BioBricks provided by iGEM Team TU Darmstadt 2014. All vectors contain a domain with a C-terminal GS-linker sequence and are flanked by an upstream BglII restriction site and a downstream BamHI restriction site. The restriction sites allow the fusion of the domains without the introduction of a restriction site between them. Here, the PCR amplified PDZ domain was digested with BglII and PstI and ligated between the restriction sites for BamHI and PstI in the SH3 vector BBa_K1497025. This allows the easy production of new scaffold BioBricks.

Figure 1: Model of a scaffold´s function. The domains are connected with a linker. They are able to build up a tight bound with enzymes assigned with a proper ligand. The educt is channeled through the enzymes and converted to the product.

The protein scaffold is an assembly platform for ligand coupled target enzymes. It was designed by the Keasling Lab in 2009 in order to improve the yield and production rate of metabolic processes. The association of target enzymes with the scaffold mimic naturally occurring catalysation cascades. In these, reaction efficiencies are optimized through the passing on of intermediates between co-located enzymes. The quick processing of intermediates can help to overcome negative production effects like unstable or toxic intermediates, metabolic bottlenecks or accumulation of undesired intermediates (Dueber et al. 2009).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1
  Illegal BamHI site found at 679
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Dueber, John E.; Wu, Gabriel C.; Malmirchegini, G. Reza; Moon, Tae Seok; Petzold, Christopher J.; Ullal, Adeeti V. et al. (2009): Synthetic protein scaffolds provide modular control over metabolic flux. In Nat. Biotechnol. 27 (8), pp. 753–759. DOI: 10.1038/nbt.1557.